Calcium Phosphate Transfection Kit Protocol

Introduction

Calcium phosphate transfection is a commonly used method for the introduction of DNA into eukaryotic cells. This technique has been used to obtain both transient¹ and stable² transfections in a wide variety of cell types. The procedure is based on slow mixing of HEPES-buffered saline containing sodium phosphate with a CaCl₂ solution containing the DNA. A DNA−calcium phosphate co-precipitate forms, which adheres to the cell surface and is taken up by the cell, presumably by endocytosis. Glycerol shock may increase the uptake of DNA in some cell types.

Reagents provided

The reagents supplied in the Calcium Phosphate Transfection Kit (Product No. CAPHOS) are sterilized by 0.2 µM filter and aseptically filled. The kit allows for either:

• 80 transfections on 10 cm dishes
• Or 160 transfections on 6 cm dishes (approximately 25 X 6 well plates)
• Or 400 transfections on 3.5 cm dishes (approximately 36 X 12 well plates)

The Calcium Phosphate Transfection Kit (Product No. CAPHOS) contains the following:

• 1 vial 2.5M CaCl₂ (Product No. C2052)
• 1 vial (25 mL) Molecular Biology grade water (Product No. W4502)
• 1 vial (25 mL) 2x HEPES Buffered Saline, pH 7.05 (Product No. H1012)
  50 mM HEPES, 280 mM NaCl, 1.5 mM Na₂HPO₄

Storage

Store all components at -20 °C.
Thaw and equilibrate all kit components to room temperature before use.

Procedure

The procedure stated below is designed for the transfection of CHO cells with 1 µg/µL pSV40-CAT plasmid (diluted in sterile molecular biology grade water). Culture cells in standard serum-containing or serum-free medium appropriate for the cell type. Antibiotics are not recommended. Use good aseptic technique and use only sterile materials.

DNA plasmids should be high-quality, ethanol-precipitated, resuspended in molecular biology grade water to a final concentration of 1 µg/µL.

This protocol can be optimized for use with a wide variety of cell types. Seeding density, amount of DNA used, incubation time and glycerol shock can easily be varied to achieve higher expression and lower toxicity when needed.

Day One: Plate Cells

Plate the cells according to the following chart:

Culture Dish	Cell Plating Density
3.5 cm dish	2 x 105
6 cm dish	5 x 105
10 cm dish	1 – 2 x 106

Day Two: Transfection

1. To prepare the cells for transfection, add fresh complete medium according to the chart below:

Culture Dish	Fresh complete medium added (mL)
3.5 cm dish	1
6 cm dish	2
10 cm dish	4

2. Two hours later, prepare two tubes with transfection reagents as follows:

Culture Dish	Tube A (mix gently)			Tube B
		CaCl2 (µL)	H2O (µL)	DNA (µL)	HeBS (µL)
3.5 cm dish	6	49	5	60
6 cm dish	15	123	12	150
10 cm dish	30	245	25	300

3. Bubble the HeBS (tube B) using an automatic pipette pump attached to a 1 mL serological pipette fitted with a 200 uL pipette tip.

4. While bubbling the HeBS (tube B), add contents of tube A (from step 2), dropwise.

5. Vortex for 2 – 4 seconds.

6. Allow the precipitate to sit undisturbed for 20 minutes.

7. Drop the solution evenly over the cell culture medium on the plate. Gently agitate the dish to distribute the precipitates evenly over the cells on the plate.

8. Incubate cells overnight (approximately 16 hours).

Day Three: Optional Glycerol Shock

1. Mix the following in a centrifuge tube:

Culture Dish	50% sterile glycerol (µL)	2x HeBS (µL)	H2O (µL)
3.5 cm dish	100	250	150
6 cm dish	225	565	335
10 cm dish	450	1130	670

2. Remove medium from the dish and replace with glycerol solution. Incubate 2 minutes.

3. Remove glycerol solution and wash twice with PBS:

Culture Dish	Volume PBS for each wash (mL)
3.5 cm dish	2
6 cm dish	5
10 cm dish	10

Day Three: Change Medium

1. Following overnight incubation (or optional glycerol shock), aspirate medium (or PBS) and replace with complete medium.
2. Incubate the cells for 48 hours.
3. Collect and lyse the cells – they are ready to be used for other applications.

References

1. Graham F, van der Eb A. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 52(2):456-467. https://doi.org/10.1016/0042-6822(73)90341-3

2. Wigler M, Pellicer A, Silverstein S, Axel R. 1978. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 14(3):725-731. https://doi.org/10.1016/0092-8674(78)90254-4